Midline-1 regulates effector T cell motility in experimental autoimmune encephalomyelitis via mTOR/microtubule pathway

Background: Effector T cell activation, migration, and proinflammatory cytokine production are crucial steps in autoimmune disorders such as multiple sclerosis (MS). While several therapeutic approaches targeting T cell activation and proinflammatory cytokines have been developed for the treatment of autoimmune diseases, there are no therapeutic agents targeting the migration of effector T cells, largely due to our limited understanding of regulatory mechanisms of T cell migration in autoimmune disease. Here we reported that midline-1 (Mid1) is a key regulator of effector T cell migration in experimental autoimmune encephalomyelitis (EAE), a widely used animal model of MS. Methods: Mid1-/- mice were generated by Crispr-Cas9 technology. T cell-specific Mid1 knockout chimeric mice were generated by adoptive transfer of Mid1-/- T cells into lymphocyte deficient Rag2-/- mice. Mice were either immunized with MOG35-55 (active EAE) or received adoptive transfer of pathogenic T cells (passive EAE) to induce EAE. In vitro Transwell® assay or in vivo footpad injection were used to assess the migration of T cells. Results: Mid1 was significantly increased in the spinal cord of wild-type (Wt) EAE mice and disruption of Mid1 in T cells markedly suppressed the development of both active and passive EAE. Transcriptomic and flow cytometric analyses revealed a marked reduction in effector T cell number in the central nervous system of Mid1-/- mice after EAE induction. Conversely, an increase in the number of T cells was observed in the draining lymph nodes of Mid1-/- mice. Mice that were adoptively transferred with pathogenic Mid1-/- T cells also exhibited milder symptoms of EAE, along with a lower T cell count in the spinal cord. Additionally, disruption of Mid1 significantly inhibited T-cell migration both in vivo and in vitro. RNA sequencing suggests a suppression in multiple inflammatory pathways in Mid1-/- mice, including mTOR signaling that plays a critical role in cell migration. Subsequent experiments confirmed the interaction between Mid1 and mTOR. Suppression of mTOR with rapamycin or microtubule spindle formation with colcemid blunted the regulatory effect of Mid1 on T cell migration. In addition, mTOR agonists MHY1485 and 3BDO restored the migratory deficit caused by Mid1 depletion. Conclusion: Our data suggests that Mid1 regulates effector T cell migration to the central nervous system via mTOR/microtubule pathway in EAE, and thus may serve as a potential therapeutic target for the treatment of MS.


Induction of passive EAE
For the induction of passive EAE, pathogenic lymphocytes were isolated from draining lymph nodes and spleen of MOG35-55-immunized Wt or Mid1 -/-mice immediately after disease onset.Cells were then cultured at the concentration of 10 7 /mL in the presence of 20 μM MOG35-55 and 20 ng/mL IL-2 (PeproTech) for 48 h.Cultured cells (5 x 10 6 /mouse) were intravenously injected into recipient mice via angular vein to induce transfer EAE as described earlier [4].

Isolation of spinal cord-infiltrating inflammatory cells
For single cell isolation, murine spinal cord tissue was cut into small pieces, then digested with 1 mg/ml type II collagenase (ThermoFisher Scientific) at 37 ℃ in a 150-rpm shaking water bath for 40 min.The resulting cells were resuspended in 40% Percoll and carefully added onto a layer of 70% Percoll and centrifuged at 500 x g to remove histiocytes and erythrocytes.The immune cells were collected from the cloudy layer at the interface between the two Percoll layers.These cells were washed with 1 x PBS buffer and utilized in the following studies.

Transwell ®
Splenocytes isolated from Wt and Mid1 -/-mice were treated with or without the presence of 3 μM rapamycin (MCE) or 100 ng/mL colcemid (Acmec biochemical) for 18 h, and then placed in the insert of a Transwell ® plate, with the lower chamber filled with 600 µL RPMI-1640 medium supplemented with 10% fetal bovine serum and 400 ng/mL C-C motif chemokine ligand 19 (CCL19, Biolegend).After 6 of incubation in a 37 ℃ CO2 incubator, cells were collected from the lower and upper chambers for cell counting and flow cytometric detection after staining with anti-mouse CD3, CD4, and CD8 antibodies, as previously described [1].For Transwell ® assay with mTOR activator treatment, Wt and Mid1 -/-splenocytes were pretreated with either 5 μM MHY1485 (MCE) or 40 μM 3BDO (MCE) for 6 h.Untreated Wt and Mid1 -/-splenocytes were stained with CellTrace™ Far-Red and CFSE, whereas treated Wt and Mid1 -/-splenocytes were labeled with 0.1 μM CellTrace™ Far-Red and 0.5 μM CellTrace™ CFSE respectively.Next, the four groups of cells with distinct fluorescent staining were mixed in equal amounts.The cells were placed into the insert of a 24-well Transwell ® plate, with the lower chamber filled with RPMI-1640 medium containing 400 ng/mL CCL-19.The cells migrated into the lower chamber were counted and analyzed by flow cytometry after 6 h of incubation in a 37 ℃ CO2 incubator.

In vivo T cells migration
Splenic T cells from Wt and Mid1 -/-mice with or without pretreatment of rapamycin (3 μM, MCE) for 18 h were labeled with CellTrace™ Far-Red or CellTrace™ CFSE (ThermoFisher Scientific).Labeled cells were then mixed at a 1:1 ratio and injected into the footpad of Wt mice.For the treatment of mTOR agonists, MHY1485 (5 μM, MCE) or 3BDO (40 μM, MCE) was added to Wt and Mid1 -/-splenocytes for 6 h.Untreated Wt and Mid1 -/-cells were fluorescently labeled with CellTrace™ Far-Red (1 μM) and CellTrace™ CFSE (5 μM) respectively, while mTOR agonist-treated Wt and Mid1 -/- splenic cells were labeled with 0.1 μM CellTrace™ Far-Red and 0.5 μM CellTrace™ CFSE respectively.The four groups of cells were then mixed in equal proportions and injected into the footpad of Wt mice.Popliteal lymph nodes were harvested 12 h after injection and the percentages of CellTrace™ Far-Red and CFSE-labeled CD3 + cells were then measured via flow cytometry.For in vivo T cell migration in the scenario of EAE, fluorescently labeled splenic cells as described above were adoptively transferred to EAE mice.The spinal cord, draining lymph nodes, and spleen were collected for the isolation of single cell suspension after 96 h, and the ratio of CellTrace™ Far-Red and CFSElabeled CD3 + cells was quantified by flow cytometry.

Histology
Histological examinations were used to assess immune infiltration and demyelination.
Spinal cords isolated from mice were embedded in paraffin after fixation.Immune infiltrations were assessed by H&E staining (Solarbio) and demyelination was examined by Luxol Fast Blue (LFB, Solarbio) staining of spinal cord sections as we previously described [2].

Cell staining
Cells were stimulated with Cell Activation Cocktail (Biolegend) for 4.5 h, followed by cell surface marker staining with indicated antibodies.Cells were then fixed and permeabilized in fixation and permeabilization buffer (Invitrogen) for 30 min, followed by staining with anti-cytokine antibodies in permeabilization buffer (Biolegend) at room temperature for 1 h.All antibodies used for flow cytometry were purchased from Biolegend or BD Biosciences.Flow cytometric detection was performed on a CytoFlex cytometer (Beckman) or ACEA NovoCyte cytometer (Agilent Technologies) and analyzed using NovoExpress software.

RT-PCR
Total RNA was extracted from the spinal cord by TRIzol Reagent (Takara) as instructed by the manufacturer.Reverse transcription of total RNA was performed using cDNA Transcriptase kit (Yeasen).The real-time PCR amplification of target genes was performed on a Quantage225 thermal cycler (Kubo Technology).The primer sequences for Gapdh, Mid1, and Cd3 genes are listed below.Gapdh, forward: GGT TGT CTC CTG CGA CTT CA; reverse: TGG TCC AGG GTT TCT TAC TCC; Mid1, forward: GGA GCT

GAC CTG TCC TAT TTG; reverse: CCG CTC ACA GAT GCT TTC T. Cd3, forward: TGG AGC AAG AAT AGG AAG GC; reverse: CAT AGT CTG GGT TGG GAA CAG.
Western blot RIPA (Yeasen) was used to extract the total proteins from mouse splenic T cells, which were subsequently separated on SDS-PAGE and transferred to a PVDF membrane (0.45 m pore size, Millipore).The membranes were blocked using a 5% skimmed milk in TBST (TBS buffer containing 0.05% TWEEN).Next, the membranes were incubated with the indicated primary antibody overnight at 4°C, followed by 5 times of washes with TBST buffer and 1 h incubation with secondary antibody (1:5000, Cell Signaling Technology) at room temperature.For primary antibodies, anti-Mid1 antibody (1:1000, abcam) and anti-β-actin antibody (1:2000, Cell Signaling Technology) were used.

RNA-Sequencing
For RNA sequencing, total RNA was sent to BGI Genomics for sequencing and analyzed using the Dr.Tom system.

Statistical analysis
All data are presented as mean ± standard error of the mean (SEM).The difference in EAE clinical score was assessed with two-way ANOVA and the EAE incidence was compared with χ 2 test using GraphPad Prism 8.The differences between the means of two groups were accomplished by unpaired Student's t-test.The differences between the means of three or more independent groups were accomplished by one-way ANOVA.
Using GraphPad Prism 8 for statistical analysis and graphing, p < 0.05 was considered statistically significant.

Figure S4 :
Figure S4: Single cells from the spinal cord or spleen of EAE mice were stained with anti-mouse CD45 and CD11b antibodies.Representative flow cytometric density plots (A) and statistical bar graphs (B) showed the proportions of CD11b + in the spinal cord of Wt and Mid1 -/-mice.Data are shown as mean ± SEM. ns, not significant.C, Bar graph showing the proportion of CD11b + in the spleen of Wt/Mid1 -/-EAE mice.Data are shown as mean ± SEM. ns, not significant.

Figure S5 :
Figure S5: Spinal cord tissues were isolated for the preparation of single cell suspension 1 week after EAE onset.Cells were then stained with anti-mouse CD45, CD3, and CD4, Representative images showing the gating of CD45 + (A) and CD3 + T cells (C).The proportions of CD45 + (B), and CD45 + CD3 + T cells (D) were analyzed.Data are shown as mean ± SEM. *, p < 0.05.

Figure S6 :
Figure S6: Wt and Mid1 -/-T cells were harvested from Wt and Mid1 -/-mice and were transferred to Rag2 -/-mice to generate chimeric mice with Wt or Mid1 -/-T cells.EAE was then induced with MOG35-55 48 h later.The mortality of mice was observed.

Figure S7 :
Figure S7: A, Single-cell suspensions were prepared from the lymph nodes of untreated Wt and Mid1 -/-mice (10-12 weeks old).The proportions of CD3, CD4, and CD8 T cells were determined by flow cytometry.Data are shown as mean ± SEM.B-C, The proportions of TH1, TH17, TC1, and TC17 were determined.Data are shown as mean ± SEM. ns, not significant.

Figure S9 :
Figure S9: Wt and Mid1 -/-splenic cells were treated with or without the presence of 5 μM MHY1485 (MCE) or 40 μM 3BDO (MCE) for 6 h, the cells were added to the insert of a 24-well Transwell ® plate, with the lower chamber filling with 1640 medium containing 400 ng/mL CCL-19.The cells migrated to the lower chamber were counted and stained with CD3, CD4 and CD8 antibodies for flow cytometric analysis after 6 h.*, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.